Hepatocyte-like cells within the pancreas of rats fed methyl-deficient diets.

Administration of a methyl-group-deficient diet with or without an initial single injection of diethylnitrosamine to male Fischer 344 rats resulted in the development of cells structurally similar to hepatocytes that lay in small clusters around one or more islets of Langerhans in the pancreas. These cells were detected in 15/256 rats fed various methyl-deficient diets in study 1 and in 7/28 rats fed a severely methyl-group-deficient diet in study 2. The results of special staining procedures for the detection of glycogen, albumin and particulate iron uptake indicated that at least some of these cells shared features common to liver cells. These results, as well as previously reported observations of similar cells in hamsters treated with ethionine, provide additional evidence that the lack of available physiological methyl donors contributes to altered differentiation of pancreatic acinar cells.

Hepatic DNA methylation and liver tumor formation in male C3H mice fed methionine- and choline-deficient diets.

The effects of the chronic administration of methyl-deficient, amino acid-defined diets on liver tumor formation were examined in male weanling C3H/HeN mice previously treated with a single ip injection of 0 or 150 mg diethylnitrosamine/kg body weight [(DENA) CAS: 55-18-5]. Five diets were used: diet 1, adequate; diet 2, devoid of both methionine and choline; diet 3, devoid of methionine only; diet 4, devoid of choline only; and diet 5, devoid of methionine, choline, folic acid, and vitamin B12. Equimolar homocystine replaced methionine in all methionine-devoid diets. All diets were administered for 1 year. No hepatocellular carcinomas and only 3 liver adenomas were seen among the 129 animals at risk in the 5 groups that had received no DENA. Among the DENA-treated groups fed diets 1-4, the incidence of hepatocellular carcinomas in the mice at risk averaged 40%, with no significant differences noted among groups. A relatively low incidence of liver carcinomas (10%) was seen among DENA-treated mice subsequently fed diet 5; it could be ascribed to the enhanced mortality seen in these animals due to the dietary deficiencies. Lung tumors were seen in 44% of the DENA-treated mice surviving more than 35 experimental weeks and in only 2.5% of the corresponding DENA-untreated animals. Feeding diet 2, deficient in methionine and choline, to male C3H mice for 5-20 weeks decreased the hepatic ratio of S-adenosylmethionine (CAS: 29908-3-0) to S-adenosylhomocysteine (979-92-0) relative to that observed in mice fed the adequate diet 1. The 5-methyldeoxycytidine [(5-MC) CAS: 838-07-3] contents of liver DNA in animals fed diet 2 for 5, 10, and 20 weeks, however, were not significantly different from the corresponding levels in diet 1-fed mice. The results indicate that a methionine- and choline-deficient dietary regimen that lowers the 5-MC levels in DNA and enhances liver tumor formation in male F344 rats does not do so in male C3H mice.

Ethionine carcinogenesis in CD-1, BALB/c and C3H mice.

In two separate in vivo studies, ethionine was evaluated for carcinogenic activity in mice. In the first study, DL-ethionine was fed in a chow diet at 0 (controls), 0.1 (low dose, LD) and 0.25% (high dose, HD) concentrations to the following groups of mice (30 animals/group): Swiss Webster CD-1 females, BALB/c males, and C3H/HeN males and females. Because of severe toxicity, BALB/c females were fed 0.05% (LD) and 0.1% (HD) ethionine. The Swiss and BALB/c mice were maintained on their respective diets for up to 105 weeks before killing whereas the C3H mice were killed at 68 weeks because of the high spontaneous incidence of liver tumors in this strain. The percentages of animals at risk (surviving the time to the first liver tumor recorded in each sex and strain) that bore liver tumors were as follows: Swiss female control, 0% (0/29), Swiss female LD, 87% (20/23); Swiss female HD, 89% (16/18); C3H male controls, 35% (8/23); C3H male LD, 55% (16/29); C3H male HD, 58% (15/26); C3H female controls, 5% (1/20); C3H female LD, 60% (12/20); C3H female HD, 92% (12/13); BALB/c male controls, 4% (1/23); BALB/c male LD, 8% (2/24); BALB/c male HD, 31% (5/16); BALB/c female controls, 0% (0/30); BALB/c female LD, 52% (14/27); and BALB/c female HD, 92% (12/13). The female mice were more responsive than the males in developing liver tumors. The results of the feeding study are compared with those obtained in a second study in which C3H female mice were intubated with 0, 150 or 500 mg DL-ethionine/kg body wt three times per week for 30 weeks and killed at 2 years. Only the LD mice showed a significantly increased incidence of liver tumors (20/39) as compared to controls (12/41) or HD mice (7/37) in the latter study. The hepatic levels of the major ethionine metabolite and methylase inhibitor, S-adenosylethionine (AdoEt), as well as of the endogenous methyl group donor, S-adenosylmethionine (AdoMet) were determined in Swiss female mice fed either 0.1 or 0.3% in the diet for 1-6 weeks. Hepatic AdoEt levels ranged from 37 to 80 micrograms/g liver in the LD animals and from 61 to 203 micrograms/g liver in the HD group; levels of the endogenous metabolite AdoMet correspondingly dropped to 65% of the normal levels. The present results (i) extend to different strains and to both sexes previous observations demonstrating the hepatocarcinogenic activity of ethionine in mice; and (ii) indicate that as in the rat such activity may be exerted through the formation of AdoEt.

Effect of methionine and choline on liver tumor promotion by phenobarbital and DDT in diethylnitrosamine-initiated rats.

The effect of the chronic feeding of methionine or choline on liver tumor promotion by phenobarbital (PhB) or 1,1 bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT) was studied in rats receiving an initiating dose of diethylnitrosamine (DEN). Male weanling rats were injected i.p. with DEN (200 mg/kg body wt). Control rats were injected with saline. Five days after the injection, the rats were placed on different diets containing 0.05% PhB or 0.05% DDT with or without added 1.5% DL-methionine or 1.0% choline chloride. Each diet was administered for 72 weeks, when the animals were placed on the unsupplemented chow diet for an additional 30 weeks. Rats treated with DEN and then fed PhB or DDT developed an 85-100% incidence of hepatocellular carcinomas (HCCs). Single injection of DEN alone produced a 60% incidence of HCCs. Dietary feeding of methionine and choline either alone or in combination with PhB or DDT did not have any significant effect on the incidence of HCC's. Liver tumor formation was negligible in uninitiated rats. Lung metastases developed in 42% and 46% of the DEN + PhB- and DEN + DDT-treated groups, respectively. Supplementation of methionine in the diet lowered the incidence of lung metastases to 14% in the DEN + PhB-treated rats and to 19% in DEN + DDT-treated rats. Choline was not effective in inhibiting the development of lung metastases in either case. The injection of DEN alone produced a 54% incidence of lung tumors. PhB and DDT feeding lowered the DEN-induced lung tumor incidence to 23% and 14% respectively. Further, when the data from different diet groups were combined it was found that single injection of DEN also doubled the incidence of leukemia normally seen in F344 rats. The present report constitutes the first evidence that a single injection of DEN induces lung tumors and enhances the incidence of leukemia in rats.

Effects of dietary calcium acetate on lead subacetate carcinogenicity in kidneys of male Sprague-Dawley rats.

Seven groups of 4-week-old male Sprague-Dawley rats, 30 animals/group, were fed for 18 months a commercial chow diet with or without 1% admixture of lead subacetate (Pb-Acet) and 0, 0.3, 1, 3 and 6% of calcium acetate (CaAcet). Feeding a 3% CaAcet-only diet did not produce any pathomorphological effects except for a slight decrease in body weights of the rats to 93% of that seen in the control rats. Feeding the 1% PbAcet-only diet decreased the growth rate of rats to 80% that of the control animals and increased the organ/body weight ratios of the kidneys and livers to 200% and 114% of the corresponding control values. Kidney tumors developed in 45% of the rats treated with PbAcet only, with the earliest appearing at 58 weeks. No tumors of other tissues were found in those rats. Feeding the PbAcet + CaAcet diets reduced the weights of rats from 74% to 54% of that of the control rats and increased the kidney/body weight ratio to 300% that of the control animals. The incidences of renal tumors increased in those rats to an average of 71% (p less than 0.03 versus the PbAcet-only diet), with no significant effects relative to the CaAcet contents in the diets. Primary non-renal tumors were found only occasionally in rats fed the PbAcet + CaAcet diets, without significant differences among the various treatment groups. The renal lead accumulation at 18 months equalled 572 +/- 102, 295 +/- 43, 247 +/- 10, 168 +/- 9, and 162 +/- 6 micrograms/g dry weight (mean +/- S.E.M.; n = 22-24) for the 0, 0.3, 1, 3 and 6% CaAcet in the PbAcet diet, respectively. The hepatic accumulation of lead was 22.2 +/- 0.7 micrograms/g dry weight with no discernible differences among the various PbAcet + CaAcet treatment groups. The results indicate that CaAcet added to the PbAcet diet increases the toxicity of the lead salt, and tends to enhance the renal carcinogenicity of PbAcet, yet decreases the accumulation of lead in the kidneys. No significant adverse effects of the PbAcet and CaAcet treatments on the livers were found.

Profound postinitiation enhancement by short-term severe methionine, choline, vitamin B12, and folate deficiency of hepatocarcinogenesis in F344 rats given a single low-dose diethylnitrosamine injection.

The potential promoting and/or complete carcinogenic activity of a methyl group-deficient (MD) diet lacking methionine, choline, vitamin B12, and folate on liver tumor induction in weanling male F344/NCr rats was examined. Each of 50 rats per group received one injection 20 mg diethylnitrosamine [(DENA) CAS: 55-18-5; N-nitrosodiethylamine]/kg body weight at 4 weeks of age, and then each was maintained on a methyl group-adequate (MA) diet for 52 weeks (groups 2 and 5) or on an MD diet for 15 weeks followed by the MA diet for 37 weeks (group 4). Controls received injections of saline and were maintained on the same two respective diet regimens (groups 1 and 3, respectively). Histologic results from sacrifices at 6, 10, 15, 22, 39, and 52 weeks revealed early development of foci of eosinophilic gamma-glutamyltransferase (GGT)-positive hepatocytes by week 6 in DENA-MD diet-treated rats, with subsequent development of a diffuse hyperplasia of hepatocytes, oval cell proliferation, cholangiofibrosis, nodular cirrhosis, and neoplastic nodule (NN) formation and, at 52 weeks, hepatocellular carcinomas (HCC) in 13 of 15 rats. Similar but significantly fewer lesions were observed at slightly later sacrifice times in the livers of saline-MD diet-treated rats, with development of NN in 5 of 12 rats and an HCC in 1 of 12 rats at 52 weeks. DENA-treated rats on MA diets developed relatively few GGT-positive foci, and none developed any neoplastic lesions. Except for basophilic foci, areas and foci of cellular alteration containing glycogen-rich hepatocytes frequently exhibited diminished uptake of injected iron and decreased glucose-6-phosphatase and ATPase contents focally or throughout. This study indicates that a relatively brief exposure of both untreated and DENA-treated weanling rats to a severely MD diet produces classical preneoplastic and neoplastic lesions in their livers.

Hyperplastic thyroid lesions in fish.

The teleost thyroid lacks a capsule, can be found ectopically in certain tissues throughout the animal, and is frequently predisposed to extensive proliferation. Several genetically susceptible small fish species as well as several larger salmonid and cyprinid species undergo a marked proliferation of the thyroid due to external conditions, such as poor water quality and abnormal nutrition, often making it difficult for one to distinguish these lesions as either cancer or goiter. The history of this controversy and present data from my study in which a marked growth of the thyroid occurred in guppies (Poecilia reticulatus) held at 27 degrees C but not in those held at 17 degrees C are reviewed. The significance of this predisposition in small fish to carcinogenicity testing is discussed.

Hepatocarcinogenesis in rats fed methyl-deficient, amino acid-defined diets.

The ability of methyl-deficient, amino acid-defined diets to produce liver tumors was studied in rats treated both with and without initiating doses of diethylnitrosamine (DENA). Male, weanling F344 rats were fed a complete, amino acid-defined diet for one week. They were then injected once i.p. with one of 3 doses of DENA (20, 70 or 200 mg/kg body weight) and fed the complete diet for an additional week. Thirty animals from each dose group were then maintained for 76 weeks on the complete diet (Diet 1) or one of 4 methyl-deficient diets: Diet 2, devoid of methionine and choline; Diet 3, devoid of methionine only; Diet 4, devoid of choline only and Diet 5, devoid of methionine, choline, folic acid and vitamin B12. In Diets 2, 3 and 5 methionine was replaced by equimolar amounts of its metabolic precursor DL-homocystine. Control rats were injected i.p. with the saline vehicle and maintained for the 76-week period on Diets 1 and 2. Forty percent of the rats fed Diet 2, but receiving no DENA, developed hepatocellular carcinomas or cholangiomas. A 90-100% incidence of hepatocellular carcinomas was seen in all groups initiated with DENA and fed Diet 2. No malignant liver tumors developed in Diet 1 rats that had received 0 or 20 mg/kg DENA; however, hepatocellular carcinomas were noted in one-half of such animals receiving the 70 and 200 mg/kg doses. Liver metastases grew in the lungs of 60% of the tumor-bearing rats fed Diet 2; none were seen in the Diet 1-fed rats. The singly deficient Diets 3 and 4 enhanced liver tumor formation to DENA-initiated rats to a significantly lesser extent than did Diet 2. All DENA-initiated rats fed the severely deficient Diet 5, died within 23 experimental weeks with livers containing hepatocytes of atypical appearance and, particularly at the 2 higher dosages, a cirrhotic pseudonodular architecture. No hepatocellular carcinomas or cholangiomas were observed in Diet 5-fed rats. None of the diets tested appeared to enhance tumor formation in extrahepatic tissues. In fact, significant decreases were noted in the formation of spontaneous testicular interstitial cell tumors in Diet 2-fed rats and of pancreatic acinar tumors in rats fed Diets 2 and 3. Diet 2, devoid of both methionine and choline, also induced metaplasia of pancreatic acinar cells to hepatocyte-like cells and was associated with moderate to severe hyperplasia of the transitional epithelium lining the renal pelvis.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of calcium and magnesium acetates on the carcinogenicity of cadmium chloride in Wistar rats.

The effects of calcium and magnesium acetates on the formation of injection site and testicular tumors in male Wistar rats over 2 years following s.c. injections of cadmium chloride (CdCl2) were determined. The rats (25/group) received a single s.c. dose of CdCl2 (0.02 or 0.04 mmol/kg; 0.9% NaCl solutions). Calcium and magnesium acetates were administered as 3% dietary supplements for 2 weeks prior to and 2 weeks after the CdCl2 injection, or as three daily s.c. injections (0.16 mmol calcium acetate per kg, 4 mmol magnesium acetate per kg; 0.9% NaCl solutions) at the same site as CdCl2 on the day before, the day of, and the day after CdCl2 dosing. Control groups were given 0.9% NaCl solution instead of CdCl2 plus s.c. or dietary calcium and magnesium acetates. In rats given injections of CdCl2 alone, the final tumor yields were 33 and 34% of rats at risk at the injection site (mostly fibrosarcomas) and 86 and 85% of rats at risk in the testes (mostly interstitial cell tumors), respectively, for the low- and high-CdCl2 doses. In control rats, the corresponding tumor yields were 0% at the site of 0.9% NaCl solution injection and 30% in the testes. Dietary calcium and magnesium acetates or s.c. calcium acetate did not affect significantly the tumor yields and latent periods. Simultaneous injections of magnesium acetate at the same site completely prevented the development of injection site tumors for both CdCl2 doses but had no effect on the final yields of testicular tumors. CdCl2 injection also caused significant elevation of incidence of the pancreatic islet cell tumors (8.5 versus 2.2%) regardless of any other experimental treatment. These results provide further evidence that the divalent carcinogenic metals may exert their activity through an antagonism with the physiologically essential divalent metals.

Visual acuity with the ITT Night Vision Aid for patients with night blindness.

The Night Vision Aid is a photomultiplier device developed by International Telephone and Telegraph Co. (ITT) and the Retinitis Pigmentosa Foundation as a mobility aid for those with night blindness. The purpose of this study was to measure visual acuity with and without the Night Vision Aid at a variety of light levels to determine how much visual assistance it provided over a wide range of illuminations. Ten normal subjects and five patients with retinitis pigmentosa (RP) used the aid at nine luminance levels ranging from 10(-6) to 10(2) ml. With the device, visual acuity improved at light levels below 0.1 ml and target visibility was extended about 3 log units further into low luminance. At light levels above 0.1 mL, unassisted visual acuity was better in all normal and most RP subjects. The best visual acuity attained with the Night Vision Aid was 6/15 (20/50). Graphs and dark adaptation curves illustrate our findings.

Quantitation of differences between spontaneous and induced liver tumors in mice with an automated image analyzer.

Spontaneous hepatocellular neoplasms of B6C3F1 mice (basophilic neoplasms) as well as those induced with the herbicide Nitrofen (eosinophilic neoplasms) were observed with an image analyzing computer to determine if quantifiable morphologic differences existed between them. Several morphologic parameters were measured on 5 liver sections from each of the following groups: (a) unexposed control liver; (b) non-trabecular basophilic neoplasms; (c) trabecular basophilic neoplasms; (d) small well circumscribed eosinophilic neoplasms; and large irregular eosinophilic neoplasms without (e) and with (f) pulmonary metastases. The total number of hepatocytes per unit area was significantly smaller in the eosinophilic neoplasms than in the basophilic neoplasms or the controls. This was the result of a greatly increased cell cross-sectional area in the eosinophilic neoplasms, caused predominantly by a larger cytoplasmic cross-sectional area. Nuclear cytoplasmic ratios of cells in eosinophilic neoplasms were lower than in the other groups for this reason. This demonstrates that quantitative morphologic differences exist between the spontaneous and induced neoplasms, which supports the conclusion that Nitrofen is a true carcinogen, and not a promoting agent.

Intranuclear inclusion bodies within neurons of spinal and cranial ganglia in three cyprinid species.

A histological examination of 205 fish representing four cyprinid species from a site 2.5 miles north of Wheeling, West Virginia, on the Ohio River revealed large (2--4 micron) cuboidal intranuclear inclusion bodies (NIB's) within neurons in the cranial and spinal ganglia of three species. Because the minnows had been caught during a yearly sampling of fish, an additional 63 minnows were taken the following year. Inclusions were again observed. The NIB's stain strongly with phloxine as well as with Mallory and Giemsa stains, appearing bright red or pink. Various histochemical tests indicated that the inclusions contain protein and lipid but no carbohydrates or nucleic acids. No heavy metals were detected by electron probe analysis. At the ultrastructural level the inclusions exhibit subunits resembling hexagons measuring 326--350 nm. Previously suggested causes for such inclusions include effects of viruses, aging, drugs, cellular transformation, and an altered metabolic state of affected cells.

Histopathologic differences between liver tumors in untreated (C57BL/6 X C3H)F1 (B6C3F1) mice and Nitrofen-fed mice.

A comparison was made of the histologic characteristics of naturally occurring and Nitrofen-induced liver tumors in (C57BL/6 X C3H)F1 (B6C3F1) mice. Whereas induced tumors generally consisted of solid sheets or nodules of frequently enlarged eosinophilic hepatocytes containing enlarged and/or hyperchromatic nuclei, the spontaneous neoplasms consisted predominantly of small basophilic cells containing oval or round nuclei and frequently arranged in trabeculae 1--2 cells wide. Within the livers of Nitrofen-treated animals, foci of hepatocytes resembling those within the tumor masses were also observed in the nonneoplastic areas. Metastases were not present in the lungs of any controls, whereas small emboli or large masses of neoplastic hepatocytes were present in incidences up to 29% in treated mice with tumors. This bioassay provides evidence that, in some cases, liver tumors in treated mice are morphologically different from those in controls and suggests that Nitrofen induces unique liver tumors rather than acts as a promoter of spontaneous neoplasms.